Process for the synthesis of 4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′, 4′, 5′7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one (Rose Bengal) and related xanthenes

ABSTRACT

A new process for the manufacture of iodinated xanthenes in high purity includes a cyclization step followed by an iodination step. No extraction, chromatographic or solvent concentration steps are required, and the intermediate as well as final compounds are isolated via filtration or similar means. The process requires a single organic solvent, and the steps are completed at temperatures below 100° C. The exclusion of chloride ions, of chloride free-radicals, hypochlorite ions, or hypochlorous acid as reagents or from reagents that may generate these species in situ in the presence of oxidants, prevents undesirable impurity formation. Several new compounds have been conceived and isolated using these methods. These new compounds are also formed into new medicaments.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation-in-part of copending U.S. applicationSer. No. 13/916,408, filed on Jun. 12, 2013 which is a divisional ofU.S. application Ser. No. 12/884,833, filed on Sep. 7, 2010 (now U.S.Pat. No. 8,530,675 issued Sep. 10, 2013) which claims benefit fromprovisional application Ser. No. 61/243,701 filed on Sep. 18, 2009,which are all incorporated herein by reference.

FIELD OF THE INVENTION

The present invention relates to methods of preparation or synthesis andisolation of highly purified Rose Bengal, Rose Bengal Lactone andrelated xanthenes, and also relates to highly purified Rose Bengal, RoseBengal Lactone and related xanthenes. One aspect of the presentinvention relates to synthetic procedures to prepare iodinatedfluorescein derivatives that contain at most 2% by weight, andpreferably less than 0.15% by weight, of individual organic impurities.Controlling impurities to the 0.15% or 1500 ppm level or lower isrelevant for pharmaceutical utility since this represents thequalification threshold for compliance with International Conference ofHarmonisation (ICH) guidelines. Hence, another aspect of this inventionrelates to the pharmaceutical utility and identification of the novelcompounds that are disclosed herein, for which the above synthesis wasspecifically engineered to control their formation.

BACKGROUND OF THE INVENTION

The fluorescein structural motif and one step cyclization from phthalicanhydride and resorcinol is believed to have been first described in1871 by Baeyer (Berichte. 1871: 4, 555). Graebe (Annalen. 1887: 18, 318)is believed to be the first to use halogenated phthalic anhydride assubstrates in the cyclization noting in his report the use of an excessof anhydride (1.3 equivalents) to resorcinol. Iodination ofdichlorofluorescein appeared in the literature in 1887 with a report byLe Royer (Annalen. 1887: 238, 359). In the 20^(th) century, several usesfor fluorescein analogs emerged. The compounds have been used as textiledyes, biological stains, building blocks for non-volatile memorydevices, thermoimaging substrates and food and cosmetics coloring. Forexample, erythrosine (FD&C No. 3) and partially iodinated erythrosine(D&C Nos. 11 and 12) are used as food, drug and cosmetic dyes. Aparticular tetraiodo-xanthene, Rose Bengal, has been used forvisualization of ocular disease and, in radiolabeled form, as a medicaldiagnostic for liver function, appearing in the United StatesPharmacopeia in 1965.

The cyclization, however, to create the xanthene core of Rose Bengal hasnot substantially improved from the 1880's technology (high temperaturemelts in open kettles), even though interest in the synthesis of thenon-halogenated analogs and elaboration on the fluorescein motif isextensive. The known synthetic methods produce a range of unpredictableand poorly characterized impurities including residual solvents,inorganic compounds and organic compounds derived from side reactions ordegradation processes. For many historical uses in industrialapplications, food dyes or diagnostics, these impurities arepermissible. For example, the United States Code of Federal Regulations(CFR) allows an impurity level for FD&C No. 3 (erythrosine) of no morethan 1% mono-iodinated impurities and no more than 9% of other loweriodinated fluoresceins. The CFR also allows residual impuritiesoriginating in the cyclization step, such as partially iodinatedphthalic acids and resorcinols (for example, see: Kamikura, ShokuhinEiseigaku Zasshi 1985: 26, 243 and Wada et al., Food. Add. Contam. 2004:21, 1137).

Such historical coloring agent specifications are quite disparate tomodern International Conference of Harmonisation (ICH) guidelines for anew drug substance, which requires reporting of impurities of 0.05% orhigher, comprehensive identification of any organic impurity present atlevels of 0.1% or higher, and thorough toxicologic qualification of anyimpurities over 0.15%, and further provide limits on inorganicimpurities and especially stringent limits on residual solvents. Hence,when introducing this class of compounds into the body at therapeuticdoses, the necessity to have a well controlled, predictable andreproducible synthesis becomes a priority. Unpredictable generation ofmultiple impurities during synthetic steps or in purification is not anoption for inclusion in such a specification, especially with apotential parenteral drug product formulation.

To make reagent grade Rose Bengal, the United States Pharmacopeia XXIIrecommends using HCl to purify Rose Bengal via an acid/basemanipulation. The present inventors have found that, quite surprisingly,when oxidants like hydrogen peroxide or oxone are present, treatment ofiodinated fluoresceins with reagents that contain or can generateaqueous chloride ions causes a side reaction where one or more of the Isubstituents can be transhalogenated to Cl. This can also occur whenchloride free radicals, hypochlorite ion, or hypochlorous acid arepresent. This side reaction during the preparation of Rose Bengal hasnot been reported previously and the cyclization step, the iodinationstep and any purification scheme must be carefully controlled to preventthis undesirable side reaction.

While iodinated fluorescein analogs have been previously described andoften generically recite the word “halogen”, none of these priordisclosures appear to enable the synthesis of iodo-xanthene substitutedfluoresceins directly from iodo-resorcinols. Also, none of the priordisclosures appear to require at least one iodine to be present in themolecule, and none claim a pharmaceutical applicability of thesecompounds that is most certainly iodine dependent. Predominant use offluoresceins for nontherapeutic purposes have resulted in a paucity ofinformation regarding the description and reduction to practice ofmethods required for the preparation of high purity activepharmaceutical ingredients in this compound class, as well as methodsfor the identification, characterization and synthesis of minorby-products which may have utility as human therapeutics or ascolorants. Iodinated xanthenes have been generically included asembodiments in various disclosures, and Rose Bengal is a well knowncompound first described in the 1880's. None of these references,however, has described the isolation or identification, nor disclosed orindicated the possibility of the existence of, the transhalogenatedminor products composed of at least one I and at least one Clsubstituent on the xanthene core. The present inventors have discoveredthat these products can exist in up to 2% by weight in commercialsamples of Rose Bengal. Furthermore, prior references allude to loweriodinated xanthene contaminants (for example, not more than 9% isallowed for the dye certification of erythrosine in the present CFR74.303), but none of these references have proposed a structure or aname for triiodinated versions that can be substituted with hydrogen ineither the 2′ or 4′ position or their corresponding atropisomers (seeFIGS. 1 q and 1 r infra and refer to U.S. Pat. No. 6,649,769 for adiscussion of atropisomerism on this scaffold). Nor has the priorreferences taught or suggested the isolation or enabled the synthesis ofindependent I/Cl substitutions about the xanthene core. The presentinvention identifies, characterizes and establishes methods toefficiently control the synthesis of these by-products to meet thestandards required for utility in pharmaceutical applications. Inaddition, the process of the present invention avoids undesirableformation of these by-products, avoids the necessity of using multiplesolvents and sets strict control of reagents, all of which improvehandling, yield, purity and applicability of the process forpharmaceutical use.

SUMMARY OF THE INVENTION

The present invention relates to materials and methods to control theimpurity level in iodinated xanthenes of Formula 3 and Formula 4manufactured for pharmaceutical use, processes for the manufacture ofthese iodinated xanthene compounds, and the disclosure of related butpreviously unanticipated transhalogenated impurities. The presentinvention also relates to materials and methods for preparing the same,and in particular processes for preparing erythrosine (FD&C 3), RoseBengal, and other related iodinated xanthenes in purity suitable forpharmaceutical use. In one embodiment, this process employs only asingle organic solvent, low temperature (less than 100° C.) and requiresuse of very limited and select additives and modifiers to avoidformation of impurities. Avoiding formation of transhalogentedimpurities can, for example, be accomplished by limiting chloride ion toless than 1500 ppm in the reaction mixtures, and especially in thereaction mixtures during the iodination step. No extraction,chromatographic or solvent concentration steps are required, and theintermediate, as well as final compounds, can be isolated via filtrationor similar means. The present invention is also directed to specificanalogs and new compounds, particularly those that can be made or areavoided via the method disclosed above. These compounds have beenisolated and identified, and are adapted for pharmaceutical, medicinal,cosmetic and colorant use.

In a further embodiment, there is provided a process for the preparationof a compound of Formula 3,

in which each position R₁ is independently C₁-C₄ alkyl, halogen or H;and R₂, R₃, R₄ and R₅ are independently I, F, Cl, C₁-C₄ alkyl or H,where at least one of R₂, R₃, R₄ or R₅ is I, and each position R₆ isindependently H or C₁-C₄ alkyl; comprising reacting a compound ofFormula 3 in which each position R₁ is independently C₁-C₄ alkyl,halogen or H; R₂, R₃, R₄ and R₅ are independently F, Cl, C₁-C₄ alkyl orH and at least one of R₂, R₃, R₄ or R₅ is H with iodine (I₂) in thepresence of aqueous base to replace at least one of R₂, R₃, R₄ or R₅with I.

Another embodiment is directed to a method of making a compound ofFormula 3 in which each position R₁ is independently C₁-C₄ alkyl,halogen or H; R₂, R₃, R₄ and R₅ are independently halogen, C₁-C₄ alkylor H; and each position R₆ is independently H or C₁-C₄ alkyl; comprisingreacting a compound of Formula 1 or Formula 1a,

in which each of position R₁ is independently halogen, C₁-C₄ alkyl or H,with a compound of Formula 2

in which R₇, R₈, R₁₀ are independently F, Cl, C₁-C₄ alkyl or H; and R₉is H.

A further embodiment is directed to a method of making derivatives ofFormula 3 in which each position R₁ is independently halogen or H; R₂,R₃, R₄ and R₅ are independently I, Br, Cl, F or H, where at least one ofR₂, R₃, R₄ and R₅ is Cl; and R₆ is H; comprising reacting the compoundof Formula 3 with a chlorine radical, chloride ion or chloride iongenerated in situ (i.e., such as a sodium hypochlorite solution, orhypochlorous acid) such that any R₁, R₂, R₃, R₄ or R₅ that is I or Brcan be independently replaced by Cl.

Another embodiment relates to compounds of Formula 3 where at least one,but no more than two, positions selected from R₂, R₃, R₄ or R₅ is Cl andeach position R₁ is independently Br, F, Cl or I; provided that any R₂,R₃, R₄ or R₅ that isn't Cl or H is I and each position R₆ isindependently H or C₁-C₄ alkyl.

The compounds of Formula 3 described herein have properties useful inthe pharmaceutical, medicinal, cosmetic and colorant industries.Accordingly, the present invention also relates to the claimed compoundsin medicaments for topical or intracorporeal application, including asan active substance in medicaments for chemotherapeutic or photodynamictreatment of human or animal disease.

The compounds of Formula 3, methods for their production, medicamentsand uses so defined herein shall include all forms of such compoundsthat have been saponified or otherwise reacted to convert such compoundsfrom their lactone form (Formula 3) to their quinoid form, (Formula 4),where R₁₁ and R₁₂ are independently H or Na, K, Li or anothercounter-ion capable of forming a salt.

In a further embodiment, the present invention relates to a method forthe preparation of a compound of Formula 4 in which R₁ is independentlyCl or Br, R₂, R₃, R₄ and R₅ are I, and R₆ is H. Combining a compound ofFormula 3a in which R₁ is independently Cl or Br, and R₂, R₃, R₄, R₅ andR₆ are H, with iodide in a solution substantially free of chloride ionsto form a compound of Formula 4 in which R₁ is independently Cl or Br,R₂, R₃, R₄ and R₅ are I, and R₆ is H, affords substantive improvementover prior methods for preparation of a compound of Formula 4, theimprovement comprising a substantial nonexistence of transhalogenatedderivatives of the compound of Formula 4 where R₁ is independently Cl orBr, at least one of R₂, R₃, R₄ and R₅ is Cl and any R₂, R₃, R₄ and R₅that is not Cl is I, and where R₆ is H.

In a further embodiment, the present invention relates to a method forthe preparation of a compound of Formula 3,

in which R₁ is independently Cl or Br; R₂, R₃, R₄ and R₅ are I; and R₆is H, comprising

Step 1: combining a compound of Formula 1 or Formula 1a,

in which R₁ is independently Cl or Br, with about two equivalents of acompound of Formula 2,

in which R₇, R₈, R₉ and R₁₀ are H, in an acidic solution;

mixing together at temperatures ranging from 20° C. to 250° C., andpreferably at a temperature of 85° C.-95° C.; and

isolating the resultant cyclized intermediate product of the Formula 3a.

in which R₁ is independently Cl or Br and R₂, R₃, R₄, R₅ and R₆ are H;followed by

Step 2: combining said intermediate of Formula 3a with achloride-ion-free aqueous solution;

treating said solution of intermediate of Formula 3a with iodine (I₂),and mixing at temperatures ranging from 20° C. to 100° C. for a timesuch that conversion to Formula 3 is substantially complete, for exampleas determined by HPLC or similar means;

quenching the reaction mixture containing Formula 3 with achloride-ion-free iodine scavenger;

acidifying said quenched reaction mixture with a chloride-ion-freeacidic solution to pH less than 5; and

isolating said final product of Formula 3.

It is further preferred in the above method for the preparation of acompound of Formula 3 that the acidic solution of Step 1 in which thecompounds of Formula 1 or Formula 1a and Formula 2 are combined is freeor substantially free of chloride ions and free or substantially free ofreagents which can produce chloride ions in the reaction mixture.

It is also further preferred in the above method for the preparation ofa compound of Formula 3 that the intermediate of Formula 3a in Step 2 isfree or substantially free of chloride ions and free or substantiallyfree of reagents or other impurities which can produce chloride ions inthe reaction mixture.

It is also further preferred in the above method for the preparation ofa compound of Formula 3 that the solution in which the intermediate ofFormula 3a and iodine are combined has a basic pH.

In a variation on the above method for the preparation of a compound ofFormula 3, Step 1 of the method for the preparation of a compound ofFormula 3 may comprise combining a compound of Formula 1 or Formula 1awith less than two equivalents of a compound of Formula 2. Thisembodiment is less preferred since the yield of Formula 3 will be loweryield than that provided using the stoichiometry of preferredembodiment.

In another further embodiment, the present invention relates to a methodfor the preparation of a compound of Formula 3,

in which R₁ is independently I, Br, Cl, F, C₁-C₄ alkyl or H, and R₂, R₃,R₄ and R₅ are independently I, F, Cl, C₁-C₄ alkyl or H, where at leastany one but no more than three of R₂, R₃, R₄ or R₅ is I, comprising:

Step 1: combining a compound of Formula 1 or Formula 1a,

in which R₁ is independently I, Br, Cl, F, C₁-C₄ alkyl or H with acompound of Formula 2

in which R₇, R₈, and R₁₀ are independently F, Cl, H, or C₁-C₄ alkyl andat least two of R₇, R₈ or R₁₀ are H and R₉ is H;

in an acidic solution free or substantially free of chloride ions andfree or substantially free of reagents or impurities which can producechloride ions in the reaction mixture;

mixing together at temperatures ranging from 20° C. to 250° C.;

isolating the resultant cyclized intermediate product of Formula 3a, forexample by filtration or similar means; followed by

Step 2: combining said intermediate of Formula 3a which is free orsubstantially free of chloride ions and free or substantially free ofimpurities or impurities which can produce chloride ions with achloride-ion-free or substantially chloride-ion-free aqueous solution;

treating said aqueous solution of intermediate of Formula 3a with iodine(I₂);

mixing at temperatures ranging from 20° C. to 100° C. for a sufficienttime such that conversion to Formula 3 is substantially complete, forexample as determined by HPLC or similar means;

quenching the reaction mixture containing Formula 3 with achloride-ion-free iodine scavenger;

acidifying said quenched reaction mixture with a chloride-ion-freeacidic solution to pH<5; and

isolating said final product by filtration or similar means.

It is preferred in the above method for the preparation of a compound ofFormula 3 that the aqueous solution of Step 2 in which the intermediateof Formula 3a and iodine are combined has a basic pH.

It is further preferred that the mixing of Step 2 be continued forsufficient time such that conversion to Formula 3 is at least 90%, andmore preferably at least 95%, and most preferably at least 98% complete,such time generally being in the range of about 1 to 24 hours, and morepreferably from about 2 to 18 hours.

In another embodiment, the present invention relates to a compound ofFormula 4 in which R₁ is independently F, Cl, Br, I, H or C₁-C₄ alkyl;R₂, R₃, R₄, and R₅ are independently Cl, H or I with at least onesubstituent selected from R₂, R₃, R₄, R₅ is I and at least one othersubstituent is Cl or H; and R₆ is independently H or C₁-C₄ alkyl; andall (a) tautomeric forms, (b) atropisomers, (c) closed lactone forms asdepicted in Formula 3, (d) enantiomers of the lactone forms depicted inFormula 3, and (e) pharmaceutically acceptable salts thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 a is an illustration of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein or RoseBengal Lactone;

FIG. 1 b is an illustration of4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein;

FIG. 1 c is an illustration of2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein;

FIG. 1 d is an illustration of4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein;

FIG. 1 e is an illustration of2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein;

FIG. 1 f is an illustration of4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein;

FIG. 1 g is an illustration of2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein;

FIG. 1 h is an illustration of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein;

FIG. 1 i is an illustration of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein;

FIG. 1 j is an illustration of4,5,6,7-tetrabromo-2′-chloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′-chloro-4,5,6,7-tetrabromo-4′,5′,7′-triiodofluorescein;

FIG. 1 k is an illustration of4,5,6,7-tetrabromo-4′-chloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4′-chloro-4,5,6,7-tetrabromo-2′,5′,7′-triiodofluorescein;

FIG. 1 l is an illustration of4,5,6,7-tetrabromo-2′,7′-dichloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,7′-dichloro-4,5,6,7-tetrabromo-4′,5′-diiodofluorescein;

FIG. 1 m is an illustration of4,5,6,7-tetrabromo-4′,5′-dichloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4′,5′-dichloro-4,5,6,7-tetrabromo-2′,7′-diiodofluorescein;

FIG. 1 n is an illustration of4,5,6,7-tetrabromo-2′,5′-dichloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,5′-dichloro-4,5,6,7-tetrabromo-4′,7′-diiodofluorescein;

FIG. 1 o is an illustration of4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein;

FIG. 1 p is an illustration of4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein;

FIG. 1 q is an illustration of an atropisomer (aR depiction) of anasymmetrically substituted xanthene; and

FIG. 1 r is an illustration of an atropisomer (aS depiction) of anasymmetrically substituted xanthene.

FIG. 1 s is an illustration of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-7′-isopropyl-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 7′-isopropyl-4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein.

FIG. 1 t is an illustration of2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoicacid disodium salt, also named4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein disodium or RoseBengal.

DETAILED DESCRIPTION OF THE PRESENTLY PREFERRED EMBODIMENTS

Definitions

“C₁-C₄ alkyl” refers to straight chain and branched saturated orunsaturated hydrocarbon groups, generally having a specified number ofcarbon atoms. Examples of C₁-C₄ alkyl groups include, withoutlimitation, methyl, ethyl, n-propyl, i-propyl, n-butyl, s-butyl, i-butyland t-butyl.

“Halo” and “halogen” may be used interchangeably, and refer to fluoro,chloro, bromo, and iodo functionalities.

“Substantially chloride free” and similar language refers to reactionconditions, reagents or intermediates that are free of chloride ion,impurities containing chloride ion, or impurities which can producechloride ion, to a level of purity sufficient to avoid undesirableformation of transhalogenated impurities of Formula 3, Formula 3a orFormula 4 at levels of 0.15% (e.g., 1500 ppm) or greater, or whereinsuch reaction conditions, reagents or intermediates contain chlorideion, impurities containing chloride ion, or impurities which can producechloride ion at a level of 1500 ppm or lower.

“Substantially free of transhalogenated impurities” and similar languagerefers to the presence of compounds of Formula 3, and Formula 4 wherein,R₁ is independently Cl or Br, at least one of R₂, R₃, R₄ and R₅ is Cland the remainder are I, and R₆ is H, at a level of 0.15% (e.g., 1500ppm) or lower.

Generic Structural Representations

The structures of various intermediate and final products of Formula 3,Formula 3a, Formula 3b and Formula 4 are illustrated for the sake ofsimplicity in their generic lactone or quinoid isomeric forms, and may,depending upon pH or other conditions, be present in their alternateisomeric faun (e.g., quinoid instead of lactone, or lactone instead ofquinoid). Such generic representation is not intended to limit thedisclosure to the specific generic isomeric forms illustrated.

Reaction Schemes

Scheme I shows one embodiment of the present invention which involves amethod of making iodinated xanthenes (such as, for example, Formula 3).This method includes reacting a phthalic anhydride (Formula 1) or adi-acid (Formula 1a) with an excess of a resorcinol (Formula 2) to givea compound of Formula 3a where R₂, R₃, R₄, R₅ and R₆ are not I and atleast one of R₂-R₅ is H. This compound, following minimal isolation, isthen subjected to iodination to give a compound of Formula 3 where atleast one of R₂, R₃, R₄ or R₅ is substituted with I.

The cyclization reaction, Step 1 above, may be carried out, for example,using neat to 10% methanesulfonic acid (MSA) in water, at temperaturesfrom 0° C. to reflux. As either Formula 1 or Formula 1a undergoesprotonation in the presence of acid, which upon dehydration gives thecommon reactive acylium ion reactive species, either Formula 1 orFormula 1a or alternatively a mixture of the same can be combined withFormula 2 in step 1 to generate Formula 3a. In a preferred embodiment,neat methanesulfonic acid is used at 85° C.-95° C., for 1-16 hours. In afurther preferred embodiment, between 2-6 volumes of MSA, morepreferably between 4 and 5 volumes of MSA, and even more preferablyapproximately 4.8 volumes of MSA, are used. Alternatively,p-toluenesulfonic acid, benzenesulfonic acid, sulfuric acid ortrifluoromethanesulfonic acid, ethanesulfonic acid, acetic acid,propionic acid, trifluoroacetic acid, camphorsulfonic acid, or an acidicsolution comprised of one or more of an alkyl sulfonic acid or arylsulfonic acid with a melting point less than about 250° C., an alkycarboxylic acid or aryl carboxylic acid with a melting point less thanabout 250° C., a non-chloride Br{acute over (ø)}nsted acid, anon-chloride or chloride-immobilized Lewis Acid, or their respectivepolymer bound or salt preparations, or aqueous solutions thereof, aloneor in combination with methanesulfonic acid, may be used to effectcyclization.

The cyclization to Formula 3a can be effected using substantiallystoichiometric amounts of reactants (e.g., with a ratio of 2:1resorcinols of Formula 2 to phthalic anhydrides or di-acids of Formula 1or Formula 1a). In a further embodiment, it may be preferable to carryout the reaction with an excess of resorcinol (e.g., from about 2.5equivalents to about 3 or more equivalents, and more preferably about3.2 or more equivalents) in order to assure complete consumption ofphthalic anhydride. After isolation of Formula 3a, the resultant solidcan be re-suspended with or without agitation, preferably with heatingto 50° C. to 70° C., to improve purity, using water and a combination ofacetone and water or DMF and water, and more preferably using acetoneand water about 60° C. Isolation and re-suspension may be repeated untilthe material of Formula 3a is of a desired purity.

The iodination reaction, Step 2 above, may be carried out using molarexcess of iodine (I₂), preferably under basic conditions. For example,the reaction may be carried out using 0.1-5 M sodium hydroxide,potassium hydroxide, sodium bicarbonate or potassium bicarbonate, attemperatures from 0° C. to reflux. In a preferred embodiment, thisreaction may be carried out using 0.4-1.0 M NaOH at 70° C.-95° C. KI,NaI or a mixture of KI and NaI can be used to solubilize iodine in thereaction mixture, for example using NaI at 1-2.5 equivalents. Reactiontimes range from 1-24 h and are dependent on the number of iodine atomsadded to the xanthene ring system.

In a further embodiment, it is possible to control the extent ofiodination by varying the reaction time, temperature, or baseconcentration. Iodine for this reaction may also be generated in situusing an oxidizing agent such as oxone or hydrogen peroxide and iodidesalts selected from potassium iodide, lithium iodide, sodium iodide orsome mixture thereof. In this particular example, it is preferable toavoid inclusion of components in the reaction mixture that could lead toundesirable transhalogenation, such as aqueous chloride, chlorine, HClor other components capable of producing halogen ions, unless anyoxidizing agent, including atmospheric oxygen and iodine, has beenremoved or quenched prior to addition of such components. For examplesodium hypochlorite is a particularly undesireable component in thisreaction mixture since it is a source of labile chloride ions and apotent oxidizer, also chloride ions, chloride radicals, otherhypochlorite derivatives, hypochlorous acid and mixtures thereof canlead to the undesireable side reaction. At reaction completion, themixture can be cooled to approximately −20° C. to 10° C., preferablybelow 10° C., and iodine can be quenched by addition of an iodinescavenger, such as sodium thiosulfate, potassium thiosulfate, ammoniumthiosulfate, potassium sulfite, sodium sulfite or a mixture thereof. Ina preferred embodiment, sodium sulfite is used to quench.

Facile isolation of Formula 3 is possible if the pH of the reaction isthen adjusted, preferably maintaining a temperature below 10° C. andusing neat aqueous sulfuric acid to 1% aqueous solution, preferably 5%sulfuric acid aqueous solution, until the pH is adjusted to between 1.5and 5, causing Formula 3 to precipitate from solution. In a preferredembodiment, the pH is adjusted to between pH 1.5 and pH 3. Afterisolation of Formula 3, the resultant solid can be re-suspended andfurther isolated at ambient temperature to remove impurities, forexample using water and a combination of acetone and water or DMF andwater, preferably acetone and water. Additionally, pH may be adjusted to5 or higher to solubilize Formula 3 as Formula 4 prior to conversionback to Formula 3 for isolation under acidic conditions.

Scheme II shows another embodiment of the present invention, whereincompounds of Formula 3 may also exist in a quinoid form (Formula 4) atsubstantially neutral or at basic pH, and these quinoids may exist assalts where one or both hydroxyl groups are replaced with a basiccounter-ion, R₁₁ and/or R₁₂, including Na, K or Li ion.

Scheme III shows a method of replacing I with Cl to generate compoundsof Formula 3b, where at least one position selected from R₂,R₃,R₄ and R₅is Cl, starting from compounds of Formula 3 where at least one positionselected from R₂,R₃,R₄ and R₅ is I.

Scheme IV shows another embodiment of the present invention, whereincompounds of Formula 4 may also be isolated as products of this process,and these quinoids may exist as salts where one or both hydroxyl groupsat R₁₁ and/or R₁₂ are replaced with a counter-ion capable of forming apharmaceutically acceptable salt, including H, Na, K or Li ion.

Preferred embodiments of the present invention relate to compounds ofFormula 3 and Formula 4 where R₁ is independently Cl or Br, and R₂-R₅are independently selected from Cl, I or H where at least 3 hydrogenatom are present at R₂-R₆ or at least one chlorine atom is present atR₂-R₅, and including atropisomers when applicable.

One set of specific embodiments of the present invention include thefollowing compounds and their pharmaceutically acceptable salts and arenamed as the lactone form (Formula 3) and the quinoid faun (Formula 4),and are illustrated in FIG. 1:

4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein, seeFIG. 1 a, and its isomeric quinoid form2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoicacid, see FIG. 1 t, commonly referred to as Rose Bengal, or in itsdisodium salt form as Rose Bengal Disodium;

4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,5′,7′-tetraiodofluorescein, see FIG.1 b, and its isomeric quinoid form2,3,4,5-tetrabromo-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,6,7-pentachloro-4′,5′,7′-triiodofluorescein, see FIG.1 c, and its isomeric quinoid forms2,3,4,5-tetrachloro-6-(2-chloro-6-hydroxy-4,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(7-chloro-6-hydroxy-2,4,5-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,4′,5,6,7-pentachloro-2′,5′,7′-triiodofluorescein, see FIG.1 d, and its isomeric quinoid forms2,3,4,5-tetrachloro-6-(4-chloro-6-hydroxy-2,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(5-chloro-6-hydroxy-2,4,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,6,7,7′-hexachloro-4′,5′-diiodofluorescein, see FIG. 1e, and its quinoid form2,3,4,5-tetrachloro-6-(2,7-dichloro-6-hydroxy-4,5-diiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,4′,5,5′,6,7-hexachloro-2′,7′-diiodofluorescein, see FIG. 1f, and its quinoid form2,3,4,5-tetrachloro-6-(4,5-dichloro-6-hydroxy-2,7-diiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,4,5,5′,6,7-hexachloro-4′,7′-diiodofluorescein, see FIG. 1g, and its isomeric quinoid focus2,3,4,5-tetrachloro-6-(2,5-dichloro-6-hydroxy-4,7-diiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(4,7-dichloro-6-hydroxy-2,5-diiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein, see FIG. 1h, and its isomeric quinoid forms2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(6-hydroxy-4,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-2′,4′,7′-triiodofluorescein, see FIG. 1i, and its isomeric quinoid forms2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(6-hydroxy-2,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-2′-chloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′-chloro-4,5,6,7-tetrabromo-4′,5′,7′-triiodofluorescein, seeFIG. 1 j, and its isomeric quinoid forms2,3,4,5-tetrabromo-6-(2-chloro-6-hydroxy-4,5,7-triiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrabromo-6-(7-chloro-6-hydroxy-2,4,5-triiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-4′-chloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4′-chloro-4,5,6,7-tetrabromo-2′,5′,7′-triiodofluorescein, seeFIG. 1 k, and its isomeric quinoid focus2,3,4,5-tetrabromo-6-(4-chloro-6-hydroxy-2,5,7-triiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrabromo-6-(5-chloro-6-hydroxy-2,4,7-triiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-2′,7′-dichloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,7′-dichloro-4,5,6,7-tetrabromo-4′,5′-diiodofluorescein,see FIG. 1 l, and its quinoid form2,3,4,5-tetrabromo-6-(2,7-dichloro-6-hydroxy-4,5-diiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-4′,5′-dichloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4′,5′-dichloro-4,5,6,7-tetrabromo-2′,7′-diiodofluorescein,see FIG. 1 m, and its quinoid form2,3,4,5-tetrabromo-6-(4,5-dichloro-6-hydroxy-2,7-diiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-2′,5′-dichloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 2′,5′-dichloro-4,5,6,7-tetrabromo-4′,7′-diiodofluorescein,see FIG. 1 n, and its isomeric quinoid forms2,3,4,5-tetrabromo-6-(2,5-dichloro-6-hydroxy-4,7-diiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrabromo-6-(4,7-dichloro-6-hydroxy-2,5-diiodo-3-oxo-9,9a-dihydro-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,5′-triiodofluorescein, see FIG. 1 o,and its isomeric quinoid forms2,3,4,5-tetrabromo-6-(6-hydroxy-2,4,5-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrabromo-6-(6-hydroxy-4,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrabromo-2′,4′,7′-triiodofluorescein, see FIG. 1 p,and its isomeric quinoid forms2,3,4,5-tetrabromo-6-(6-hydroxy-2,4,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid or isomer2,3,4,5-tetrabromo-6-(6-hydroxy-2,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid;

the aR form, see FIG. 1 q, and aS form, see FIG. 1 r, of theatropisomers possible whenever one of these compounds is notsymmetrically substituted on the xanthene core; and

4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-7′-isopropyl-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,also named 4,5,6,7-tetrachloro-7′-isopropyl-2′,4′,5′-triiodofluorescein,see FIG. 1 s, and its isomeric quinoid forms2,3,4,5-tetrachloro-6-(7-isopropyl-6-hydroxy-2,4,5-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid and2,3,4,5-tetrachloro-6-(2-isopropyl-6-hydroxy-4,5,7-triiodo-3-oxo-3H-xanthen-9-yl)benzoicacid.

Compounds of Formula 3 or Formula 4 of the present invention may containchiral centers and therefore may exist in different enantiomeric anddiastereomeric forms. The present invention relates to all opticalisomers and all stereoisomers of compounds of Formula 3 or Formula 4,both as racemic mixtures and as individual enantiomers, anddiasteroisomers of such compounds, and mixtures thereof, and to allpharmaceutical compositions and methods of treatment defined herein thatcontain or employ them, respectively. Individual isomers can be obtainedby known methods, such as optical resolution, fractionalcrystallization, optically selective reaction conditions orchromatographic separation in the preparation of the final product orits intermediate.

Likewise, the quinoid form of compounds of Formula 3 or Formula 4 wherethe xanthene ring is not symmetric due to dissimilar substituents on thecorresponding xanthene aryl rings (i.e., for example, R₂≠R₅ when R₃=R₄or R₃≠R₄ when R₂=R₅, such as the quinoid forms of compounds depicted inFIGS. 1 c, 1 d, 1 g, 1 h, 1 i, 1 j, 1 k, 1 o, 1 p) may exist as stableatropisomers (as illustrated in FIGS. 1 q and 1 r). The processdescribed herein encompasses both the minimization of the relativeamount of racemic atropisomeric pairs when they may occur as reactionimpurities as well as the preparation of these atropisomers as racemicmixtures.

In so far as the compounds of Formula 3 are acidic compounds, they arecapable of forming a wide variety of different salts with variousinorganic and organic bases. The base addition salts of the acidiccompounds of the present invention are readily prepared by treating thelactone of Formula 3 with at least one or two equivalents of the chosenmineral or organic base in an aqueous or suitable organic solvent, suchas ethanol or methanol. Upon evaporation of the solvent, filtration orusing directly an aqueous solution of the resulting salt, the desiredsalt is readily obtained in the quinoid form as described by Formula 4.Pharmaceutically acceptable salts include, for example, those formedwith sodium, calcium, potassium, magnesium, meglumine, ammonium,aluminum, zinc, piperazine, tromethamin, lithium, choline, diethylamine,4-phenylcyclohexylamine and benzathine.

The present invention also includes isotopically labeled compounds,which are identical to those of Formula 3 and Formula 4, except that oneor more atoms are replaced by an atom having an atomic mass or massnumber different from the atomic mass or mass number usually found innature. Examples of isotopes that can be incorporated into compounds ofthe present invention include isotopes of hydrogen, carbon, oxygen,fluorine, chlorine and iodine, such as ²H, ³H, ¹⁴C, ¹³C, ¹⁰C, ¹¹C, ¹³O,¹⁴O, ¹⁵O, ¹⁸O, ¹⁷O, ¹⁷F, ¹⁸F, ³²Cl, ³³Cl, ³⁴Cl, ³⁶Cl, ⁷⁴Br, ⁷⁵Br, ⁷⁶Br,⁷⁷Br, ¹¹⁷I, ¹¹⁸I, ¹²⁰I, ¹²¹I, ¹²²I, ¹²⁴I, ¹²⁶I, ¹²⁸I and ¹³¹I,respectively. Compounds of the present invention, prodrugs thereof, andpharmaceutically acceptable salts of said compounds or of said prodrugswhich contain the aforementioned isotopes and/or other isotopes of otheratoms are within the scope of the present invention. Certainisotopically labeled compounds of the present invention are useful asdiagnostic agents in drug/or substrate tissue distribution assays.Isotopically labeled compounds of Formula 3 and Formula 4 and prodrugsthereof can generally be prepared by carrying out the proceduresdisclosed in the schemes and/or examples below, but substituting areadily available isotopically labeled reagent for a non-isotopicallylabeled reagent, i.e. ¹³¹I for non-isotopically labeled iodine.

One embodiment of the present invention is where the compound of Formula4 comprises about 0.001% and less than about 20% by weight in amedicament.

One embodiment of the present invention is directed to medicaments andcertain medical uses of such medicaments, and methods for treatmentusing such medicaments, for treatment of disease of human or animaltissue, wherein a primary active component of such medicaments is ahalogenated xanthene of Formula 3 or Formula 4. Such medicaments mayfunction, for example, chemotherapeutically, as chemoablative agents, oras photodynamic agents, and are useful for the treatment of a variety ofconditions affecting the skin and related organs, the mouth anddigestive tract and related organs, the urinary and reproductive tractsand related organs, the respiratory tract and related organs, thecirculatory system and related organs, the head and neck, the endocrineand lymphoreticular systems and related organs, various other tissues,such as connective tissues and various tissue surfaces exposed duringsurgery, as well as various tissues exhibiting microbial, viral, fungalor parasitic infection.

These medicaments are available in various formulations that may includeliquid, semisolid, solid or aerosol delivery vehicles, and are suitablefor intracorporeal administration via various conventional modes androutes, including intravenous injection (i.v.), intraperitonealinjection (i.p.), intramuscular injection intracranial injection (i.c.),intratumoral injection (i.t.), intralesional injection (i.l.),intraepithelial injection (i.e.), transcutaneous delivery (t.c.), andper oesophageal (p.o.) administration. Additionally, such medicamentsare suitable for topical administration via various conventional modesand routes, including topical application directly to or proximal tocertain tissues. The active ingredients in such medicaments produce adesirable therapeutic response, such as destruction of microbialinfection, reduction or elimination of tissue irritation orinflammation, reduction or elimination of hyperproliferative tissue,reduction or elimination of cancerous or precancerous tissue, reductionor elimination of surface or subsurface lipocytes or lipid deposits, andmany other similar indications.

In a preferred embodiment, such medicaments are produced in variousformulations including liquid, semisolid, solid or aerosol deliveryvehicles, as well as in tablet, capsule, suppository, and other similarforms.

In another preferred embodiment, at least one targeting moiety iscoupled to the halogenated xanthene, of Formula 3 or Formula 4, at anyof positions R₁ to R₆ or via attachment at a hydroxyl or carbonyl group.Such targeting moieties may be selected from the group including but notlimited to deoxyribonucleic acid (DNA), ribonucleic acid (RNA), aminoacids, proteins, antibodies, ligands, haptens, carbohydrate receptors,carbohydrate complexing agents, lipid receptors, lipid complexingagents, protein receptors, protein complexing agents, chelators,encapsulating vehicles, short-chain aliphatic hydrocarbons, long-chainaliphatic hydrocarbons, aromatic hydrocarbons, aldehydes, ketones,alcohols, esters, amides, amines, nitriles, azides, hydrophilic moietiesand hydrophobic moieties.

In another preferred embodiment, the compounds of Formula 4 can be usedin the manufacture of a medicament.

EXAMPLES

The following examples are intended to be illustrative and non-limiting,and represent specific embodiments of the present invention.

General Methods

All reactions were carried out in open vessels. The vessels weresometimes covered to protect from ambient light. All reactions werecarried out under nitrogen, argon or other inert atmosphere unlessotherwise noted. All solvents and reagents used were from commercialsources, and no further purification was performed. Reactions weremonitored using high-pressure liquid chromatography (HPLC) using eithera Agilent 1100 Series Quaternary pump/variable wavelength detector (225nm detection wavelength) with Supelco Ascentis Express C18 column(4.6×150 mm, 2.7 μm at 40° C., MeCN/0.5% H₃PO₄ in H₂O, 65/35 to 90/10gradient running over 30 min) or the same instrument using a WatersSymmetry Shield RP-18 column (4.6×150 mm, 5 μm at 40° C., MeCN/10 mMK₃PO₄ pH 3 in H₂O+5% MeCN, 10/90 to 80/20 gradient over 25 min or 65/35isocratic); mass spectrometry (Agilent LC/MSD trap or Waters LCMS withsimilar chromatography conditions as HPLC, modified with formic acid);and/or thin-layer chromatography (TLC) using UV light and a iodine stainfor visualizing. Supercritical fluid chromatography (SFC) was performedusing a Thar SFC 80 with a RegisCell column (3×25 cm), loading 20-22mg/injection at a 4.5 mg/mL concentration in ethanol. Proton nuclearmagnetic resonance (¹H NMR) spectra were recorded at 300 MHz on a VarianNOVA 300 or Varian Gemini 2000 or at 400 MHz on a Varian Oxford 400,each using TMS as an internal standard. Chemical shifts are reported (asδ units in parts per million, ppm) relative to the singlet at 2.50 ppmfor DMSO-d₆ referenced to tetramethylsilane (TMS) at 0 ppm. Couplingconstants (J) are reported in Hertz (Hz). Noise-decoupled Carbon-13nuclear magnetic resonance (¹³C NMR) spectra were recorded at 75 MHz oneither a Varian NOVA 300, at 100 MHz on a Varian Oxford 400, or at 75MHz on a Varian Gemini 2000 spectrometer. Chemical shifts are reportedas δ in ppm relative to the center line of the septet at 39.5 ppm forDMSO-d₆. UV-VIS spectroscopic data were obtained on a Hitachi U-2810Double Beam Spectrophotometer or on a Spectronic Genesys 2Spectrophotometer scanning from 200-600 nm, slit width 1.5 nm, pathlength 10.0 mm

Example 1 Preparation of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(Rose Bengal Lactone, FIG. 1 a)

Step 1: Cyclization. A 500 mL round bottom flask was equipped with aheating mantle, J-Kem thermocouple, large magnetic stir bar, nitrogeninlet line, and a magnetic stir plate. This apparatus was charged withtetrachlorophthalic anhydride (1.00 eq, 50.00 g, 174.9 mmol), resorcinol(2.10 eq, 40.44 g, 367.3 mmol), and neat methanesulfonic acid (250 mL).The resulting reaction mixture was a suspension at room temperature. Thereaction mixture was purged with nitrogen and heated to 90° C. to give adark red-orange solution. The reaction was held at 90° C. for 5 hours,and then an additional 5.78 g of resorcinol (0.3 eq, 52.47 mmol) wasadded. The reaction continued to stir at 90° C. for 2.5 hours. Thereaction is deemed to be complete when the amount of residualtetrachlorophthalic anhydride is <1.0% by HPLC. A 1 L round-bottomedflask (3 neck) equipped with a mechanical stirrer, ice bath and J-Kemthermocouple was charged with ambient USP water (500 mL). The 90° C.reaction mixture was added slowly via transfer line to the USP water at<10° C. using a positive pressure of nitrogen. During the transfer ofthe reaction mixture to water, the transfer rate was controlled suchthat the temperature of the water quench mixture did not exceed 60° C.An additional 100 mL of USP water was used to rinse the reactor thentransferred to the reactor containing the water. The resultantgreen-brown suspension was allowed to gradually cool to room temperature(RT) and stirred at RT for an additional 30 min. The solids wereisolated via vacuum filtration. The reactor was rinsed with USP water(2×250 mL aliquots) and these rinses were used to rinse the wetcake. Thewetcake was pulled dry for 60 min, and then dried in vacuo at 70° C.overnight to give 88.60 g of greenish-brown solids. This material, alongwith 550 mL of acetone was added to a 2 L round bottom flask equippedwith a mechanical stirrer, J-Kem thermocouple, heating mantle, nitrogeninlet line, and a Y-adapter with a reflux condenser. The resultingsuspension was heated to reflux for 1.5 hours, whereupon it was treatedwith 530 mL of USP water (slowly added via an addition funnel over 60min) such that the temperature remained ≧56° C. During the addition ofthe water, the temperature was observed to rise (maximum temp achievedwas 62° C.). After the addition of the water was complete, theyellowish-brown suspension was held at reflux for 3 hours, and thengradually cooled to RT over approximately 30-40 minutes. The mixture wasstirred at room temperature for an additional 30 min, and then theyellowish-brown suspension was collected via vacuum filtration. Thereactor was rinsed with 50% aqueous acetone (v/v, 4×100 mL), and theserinses were used to rinse the wetcake. The wetcake was allowed to pulldry over 48 h and then further dried in vacuo at 70° C. overnight togive 74.43 g (90.5% yield at 96.8% AUC purity) of tetrachlorofluoresceinas a yellow brown solid. ¹H NMR (300 MHz; DMSO d₆) δ 10.23 (S, 2H), 6.95(d, J=8.7 Hz, 2H), 6.69 (d, J=2.1 Hz, 2H), 6.57 (d of d, J=8.7 Hz, 2H).

Step 2: Iodination. A 500 mL round bottom flask (3-neck) was equippedwith a reflux condenser, Y-adapter, J-Kem thermocouple, mechanicalstirrer, heating mantle, and a nitrogen inlet line. The reactor waspurged with nitrogen and covered with aluminum foil. This was chargedwith 10.00 grains of tetrachlorofluorescein (21.3 mmol), 30 mL of 5 MNaOH solution and 300 mL of USP water to give a dark red solution. Then,7.03 grams of sodium iodide (46.9 mmol) and 32.4 g of iodine (127.7mmol) were charged to the reactor. The reaction mixture was allowed tostir at ambient temperature for 30 min, then it was heated to 90° C.HPLC analysis of an aliquot of the reaction mixture indicated completeconsumption of starting material, no partially iodinated intermediates,and complete conversion to the desired product. After the reaction hadbeen heated at 90° C. for 1.5 hours, the heat was turned off, and thereaction mixture was allowed to gradually cool to room temperature over1.5 hours. The dark purplish-pink reaction mixture was cooled to <10° C.with an ice bath. The pH of the reaction mixture was 7.13. Sodiumsulfite (6.70 g) was added to the reaction mixture in small portions. 75mL of acetone was charged to the reactor at <10° C., and the mixture wasallowed to stir for 10 min at <10° C. While at <10° C., 5% aqueous H₂SO₄solution (24 mL) was added dropwise to achieve pH 2.03 and yield a pinksuspension. This reaction suspension was collected via vacuumfiltration. The reactor was rinsed with 25% aqueous acetone (v/v, 4×100mL) and the rinses were utilized to rinse the wetcake. The wetcake waspulled dry for 3 hours and dried in vacuo at 60° C. overnight to give25.81 g of pink solids. These solids and 225 mL of acetone were added toa 1 L round bottom flask (3 neck) equipped with a mechanical stirrer,J-Kem thermocouple, and Y-adapter with nitrogen inlet. This mixture wasstirred at room temperature for 10 min, then 255 mL of USP water wasadded over 10 min to give a suspension. The suspension was stirred atroom temperature for 2.25 hours, and then filtered via vacuum filtrationto isolate solids. The reactor was rinsed using 50% aqueous acetone(1×75 mL), and this was used to rinse the wetcake. The wetcake was thenrinsed with 50% aqueous acetone (2×75 mL) and USP water (1×75 mL),pulled dry for 1 hour and further dried at 80° C. to give 18.68 g ofproduct (90.2% yield, coral pink solids, HPLC AUC purity of 99.5%) of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one.

¹H NMR (300 MHz; DMSO d₆) δ 10.27 (s, 2H), 7.59 (s, 2H). ¹³C NMR (300MHz; DMSO d₆) δ 163.2, 158.6, 151.9, 146.7, 138.8, 136.5, 135.3, 131.2,126.8, 124.6, 110.8, 81.8, 79.3, 77.0. MS MSD Trap: m/z 974.8 (M+1)⁺(exact mass 973.67). UV-VIS λmax=557 nm in methanol; melting point (mp)determined by differential scanning calorimetry (DSC)=215° C.

Example 2 Preparation of4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 b)

Step 1: Treatment of tetrabromophthalic anhydride (28.63 g, 61.74 mmol)and resorcinol (17 g, 154.4 mmol) in 143 mL methanesulfonic acidaccording to the procedure described in Example 1 Step 1 provided 33.64g of tetrabromofluorescein was isolated as a light beige solid (84.1%yield, AUC purity of 97.4%). ¹H NMR (300 MHz; DMSO d₆) δ 10.17 (s, 2H),6.86 (d, J=8.64 Hz, 2H), 6.65 (d, J=2.34 Hz, 2H), 6.54 (d of d, J=8.7Hz, 2H).

Step 2: Treatment of tetrabromofluorescein (22 g, 33.96 mmol), iodine(51.71 g, 203.7 mmol) and sodium iodide (11.22 g, 74.85 mmol) accordingto the procedure described in Example 1 Step 2 provided 36.02 g (31.2mmol, 92.1% yield, AUC purity of 96.3%) of4,5,6,7-tetrabromo-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was isolated as a light pink solid. ¹H NMR (300 MHz; DMSO d₆) δ10.22 (s, 2H), 7.49 (s, 2H). ¹³C NMR (300 MHz; DMSO d₆) δ 163.9, 158.8,152.5, 149.9, 137.3, 136.7, 133.5, 127.5, 124.6, 121.1, 111.5, 82.0,80.3, 77.4. MS MSD Trap: m/z 1152.6 (M+1)⁺ (exact mass 1151.48). UV-VISλmax=558 nm in methanol, mp (determined by DSC)=227° C.

Example 3 Preparation of2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 c)

Step 1: Treatment of tetrachlorophthalic anhydride (5 g, 17.48 mmol),4-chlororesorcinol (2.78 g, 19.23 mmol) and resorcinol (2.12 g, 19.23mmol) according to the procedure described in Example 1 Step 1 provided7.24 g of 18.6% pure2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was collected and taken to the next step as a mixture.

Step 2:2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(7.24 g, 15.4 mmol) was iodinated according to the procedure describedin Example 1, Step 2. Crude product (5.47 g) was then isolated via SFCpurification (80 g/min total flow, 40% co-solvent (EtOH/0.5% TFA) inCO₂, 140 bar, 254 nm on a 3×25 cm 5 m RegisPack column) to give 427 mg(0.54 mmol, 34.7% yield) of2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one,a light pink solid in 95.8% AUC HPLC purity. ¹H NMR (300 MHz; DMSO d₆) δ10.85 (s, 1H), 10.2 (s, 1H), 7.60 (s, 1H), 7.38 (s, 1H). ¹³C NMR (300MHz; DMSO d₆) δ 163.4, 159.0, 155.8, 152.3, 151.2, 146.9, 139.1, 136.9,135.7, 131.6, 128.2, 127.1, 125.1, 116.9, 111.0, 109.7, 82.3, 80.0,78.4, 78.4, 77.5. MS MSD Trap: m/z 882.7 (M+1)⁺ (exact mass 882.2).UV-VIS λmax=554 nm in methanol.

Example 4 Degradation of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneto4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneand4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIGS. 1 a, 1 d and 1 f)

To 100 mg (0.10 mmol), of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onewas added 1.0 mL of acetonitrile and 2.0 mL of 12.5% aqueous sodiumhypochlorite at room temperature. The reaction mixture was allowed tostir at RT for 1 hour. HPLC analysis indicated two new impurities at8.38 min and 9.93 min retention time (27.8% and 45.6%, respectively),with 26.6% of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneremaining unreacted. Mass spectral analysis showed M-1 ions: M-1=791.7corresponding to a hexachloro impurity, M-1=881.4 corresponding to apentachloro impurity, and M-1=973.2 corresponding to Rose Bengallactone. The transhalogenated compounds were made individually below(see examples 5 and 7), to confirm the structural assignments andcorrelated to the products of this example using HPLC.

Example 5 Preparation of4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 d)

Step 1: Tetrachlorophthalic anhydride (2.5 g, 8.74 mmol),2-chlororesorcinol (1.33 g, 9.18 mmol) and resorcinol (1.01 g, 9.18mmol) were combined with 12.5 mL of neat methanesulfonic acid and heatedto 90° C. for 19 h then to 97° C. for 10 h whereupon the hot mixture wascarefully added to 25 mL of ice water. This suspension was extractedinto ethyl acetate and washed with water and brine, then dried oversodium sulfate. The product was isolated via a silica gel plug using66%:14%:18%:4% toluene:dioxane:hexane:acetic acid as the eluent. 1.95 gof 46.4% pure4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onewas collected and taken to the next step without further purification.

Step 2:4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(1.95 g, 4.15 mmol) was iodinated according to the procedure describedin Example 1, Step 2. Crude yield, 2.70 g of a mixture. Product wasisolated via SFC purification (80 g/min total flow, 40% co-solvent IPAin CO₂, 140 bar, 254 nm on a 3×25 cm 5 m RegisPack column) to give 630mg (0.7 mmol, 38.5% yield, 98.7% AUC purity) of4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneas a pink solid. ¹H NMR (300 MHz; DMSO d₆) δ 11.05 (s, 1H), 10.3 (s,1H), 8.3 (s, 1H), 7.55 (d, 2H). ¹³C NMR (300 MHz; DMSO d₆) δ 163.2,158.6, 154.7, 150.7, 148.0, 146.7, 138.7, 136.5, 135.3, 134.5, 131.3,126.8, 124.6, 110.5, 110.4, 82.4, 82.1, 79.3, 77.6. MS MSD Trap: m/z882.7 (M+1)⁺ (exact mass 882.2). UV-VIS λmax=555 nm in methanol

Example 6 Preparation of2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 e)

Step 1: Treatment of tetrachlorophthalic anhydride (5 g, 17.49 mmol) and4-chlororesorcinol (6.32 g, 43.73 mmol), according to the proceduredescribed in Example 1 Step 1, provided 10.31 g of crude product.Suspension of a 9.25 g portion of this in DMF:water (1:1) followed byfiltration, provided 9.70 g (2:1 DMF complex 17.16 mmol, 92.3% yield) of2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was isolated as an orange solid. ¹H NMR (300 MHz; DMSO d₆) δ 11.13(S, 2H), 7.31 (s, 2H), 6.89 (s, 2H).

Step 2: Treatment of2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(2 g, 3.71 mmol) according to the procedure described in Example 1, Step2 provided 2.56 g (3.22 mmol, 87.3% yield) of2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was isolated as a pink-orange solid in 97.9% AUC HPLC purity. ¹HNMR (300 MHz; DMSO d₆) δ 10.90 (s, 2H), 7.48 (s, 2H). ¹³C NMR (300 MHz;DMSO d₆) δ 163.2, 154.7, 147.1, 146.8, 138.7, 135.3, 134.5, 131.3,126.7, 124.5, 110.3, 107.7, 82.6, 79.0. MS MSD Trap: m/z 791.0 (M+1)⁺(exact mass 790.77). UV-VIS λmax=551 nm in methanol.

Example 7 Preparation of4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 f)

Step 1: Treatment of tetrachlorophthalic anhydride (5 g, 17.49 mmol) and2-chlororesorcinol (7.58 g, 41.98 mmol) according to the proceduredescribed in Example 1, Step 1, provided 9.3 g of the crude product.Suspension of 8 g of the crude 9.3 g in DMF:water (1:1), followed byfiltration provided 8.65 g (16.05 mmol, 91.7% AUC yield) of4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was isolated as an orange solid. ¹H NMR (300 MHz; DMSO d₆) δ 11.09(S, 2H), 7.03 (d, J=9 Hz, 2H), 6.81 (d, J=8.7 Hz, 2H).

Step 2: Treatment of4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(2 g, 3.71 mmol) according to the procedure described in Example 1, Step2 provided 2.20 g (2.78 mmol, 75.1% yield) of4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onethat was isolated as a pink-orange solid at 97.7% AUC HPLC purity. ¹HNMR (300 MHz; DMSO d₆) δ 11.05 (s, 2H), 7.58 (s, 2H). ¹³C NMR (300 MHz;DMSO d₆) δ 163.2, 154.7, 147.1, 146.8, 138.7, 135.3, 134.5, 131.3,126.7, 124.5, 110.3, 107.7, 82.8, 79.0. MS MSD Trap: m/z 791.0 (M+1)⁺(exact mass 790.77). UV-VIS λmax=554 nm in methanol.

Example 8 Preparation of2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIG. 1 g)

Step 1: Treatment of tetrachlorophthalic anhydride (5 g, 17.48 mmol),4-chlororesorcinol (2.78 g, 19.23 mmol) and 2-chlororesorcinol (2.78 g,19.23 mmol) according to the procedure described in Example 1, Step 1provided 6.2 g of crude product (approximately 54% pure by HPLC2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one)that was collected and taken to the next step as a mixture.

Step 2:2′,4,5,5′,6,7-pentachloro-3′,6′-dihydroxy-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(6.2 g, 54.4% desired compound, 6.25 mmol) was iodinated according tothe procedure described in Example 1, Step 2, yielding 7.16 g (60.8%desired compound, 5.50 mmol, 88% yield). Crude product (3.37 g, 60.8%desired compound, 2.59 mmol) was isolated via SFC purification (80 g/mintotal flow, 40% co-solvent (50/50 IPA/EtOH 0.5% TFA in CO₂, 140 bar, 254nm on a 3×25 cm 5 m RegisPack column) to give 427 mg (0.54 mmol, 20.8%recovery from SFC) of2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneas a light pink solid. ¹H NMR (300 MHz; DMSO d₆) δ 11.05 (s, 1H), 10.85(s, 1H), 7.60 (s, 1H), 7.40 (s, 1H). ¹³C NMR (300 MHz; DMSO d₆) δ 163.1,155.4, 154.7, 149.6, 148.0, 146.5, 138.7, 135.3, 134.5, 131.3, 127.9,126.8, 124.7, 116.7, 110.3, 109.0, 107.5, 82.4, 79.6, 78.5. MS MSD Trap:m/z 790.7 (M+1)⁺ (exact mass 790.7). UV-VIS λmax=552 nm in methanol.

Example 9 Degradation of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneto4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIGS. 1 a and 1 i)

To 6.5 g (6.67 mmol) of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onewas added 130 mL acetone, 30 mL of water and 6.6 g sodium iodide (44.0mmol). The mixture was heated to reflux for 82.5 h and HPLC analysisindicated 35.4% of the title compound was present in a mixture withstarting material. The reaction mixture was diluted with 130 mL of waterand 150 mL of ethyl acetate and allowed to stand overnight at roomtemperature. The organic layer was removed, and the aqueous layer wasextracted with 100 mL ethyl acetate. The combined organic layers werewashed with brine and dried over sodium sulfate, yielding 6.73 g crudeproduct containing 35.4% of the title compound by HPLC. The titlecompound was isolated via silica gel chromatography from a portion ofthis material using 66%:14%:18%:4% toluene: dioxane:hexane:acetic acid.After successive chromatography steps,4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onewas isolated as a red solid (90 mg). ¹H NMR (300 MHz; DMSO d₆) δ 11.26,(s, 1H), 10.19 (s, 1H), 7.56 (s, 1H), 7.51 (s, 1H), 6.84 (s, 1H). ¹³CNMR (300 MHz; DMSO d₆) δ 163.4, 159.0, 158.3, 151.8, 150.8, 147.2,138.6, 136.9, 135.1, 126.7, 124.5, 110.6, 108.8, 101.7, 81.2, 88.7,77.1. MS MSD Trap: m/z 848.8 (M+1)⁺ (exact mass 847.7). UV-VIS λmax=539nm in methanol.

Example 10 Preparation of4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein (FIG. 1 h) and relatedcompounds by incomplete iodination

A 500 mL round bottom flask was charged with 10 g (21.3 mmol)tetrachlorofluorescein, 13 mL 5 M NaOH, 300 mL water, 7.08 g NaI (47.2mmol) and 16.24 g iodine (64 mmol). The solution was heated to 50° C.for 6.5 h before it was cooled to room temperature. Sodium hydrogensulfite was added followed by acetic acid until the pH was 3.7. Themixture was extracted with ethyl acetate, washed with water then brineand dried over sodium sulfate, filtered and concentrated in vacuo. 18.2g of a reddish-orange foam was isolated as a mixture of 46%4,5,6,7-tetrachloro-4′,5′-diiodofluorescein and 43% of the titlecompound, 4,5,6,7-tetrachloro-2′,4′,5′-triiodofluorescein by AUC HPLCpurity. LCMS MS Scan 800-1000: m/z 846.52 (M−1)⁻ (exact mass 847.7).UV-VIS λmax=540 nm in PBS. In comparison to example 1 where 6equivalents of iodine at 90° C. yields substantially quantitativeconversion to the tetraiodinated product, this reaction using milderheating and 3 equivalents of iodine yields a mixture of lower iodinatedimpurities. This demonstrates the utility of varying reaction conditionsto control the yield of lower iodinated products.

Example 11 Degradation of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneto4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-7′-isopropyl-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one(FIGS. 1 a and 1 s)

To a round bottom flask charged with ZnCl₂ (0.5 mL, 0.50 mmol),isopropylmagnesium chloride (0.23 mL, 0.45 mmol), and Bis(PPh₃)₂PdCl₂(catalytic amount) in THF at room temperature, 200 mg (0.21 mmol)4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onein 2 mL THF was added. The solution was stirred at room temperature for38 h. The reaction was quenched with 5 mL of USP water, 0.5 mL aceticacid and 2 drops of 5M sulfuric acid. The organic layer was washed withUSP water (3 mL), dried over sodium sulfate and concentrated in vacuo.195 mg of an orange-red solid was isolated as a mixture of 2.3% of thetitle compound and 42.9% of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′,7′-tetraiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-oneby HPLC. To confirm the structure, an isolation of the title compoundwas undertaken in the following example.

Example 12 Isolation of4,5,6,7-tetrachloro-3′,6′-dihydroxy-2′,4′,5′-triiodo-7′-isopropyl-3H-spiro[isobenzofuran-1,9′-xanthen]-3-onefrom2,3,4,5-tetrachloro-6-(6-hydroxy-2,4,5,7-tetraiodo-3-oxo-3H-xanthen-9-yl)benzoicacid disodium salt (FIGS. 1 s from 1 t)

118 g (0.11 mol) of commercial grade Rose Bengal disodium salt wasdissolved in 985 mL of USP water and acidified to pH 1-2 with 400 mL of1 M HCl to give a slurry. The slurry was extracted with 1800 mL of ethylacetate and the organic layer separated. The aqueous layer was extractedwith an additional 100 mL of ethyl acetate and the organic layerseparated. The combined organic layers were concentrated until about1800 mL of solvent remained. The slurry was then treated with 500 mL ofheptane, filtered and the wetcake rinsed with 250 mL of heptane. Thesolids were dried at 60° C. and then combined with 760 mL of dioxane ina foil-covered flask under nitrogen atmosphere. The resulting slurry washeated to 95° C. and held at temperature for 70 min. The slurry wascooled and filtered. Under nitrogen, the wet cake was rinsed with 2volumes of dioxane and dried. This sample was dissolved in 45 mL of THF(inhibitor-free), concentrated to 15-20 mL and loaded onto an aluminacolumn (510 g neutral alumina with 300 mL of a 90 Acetonitrile: 10Isopropanol:1 Acetic Acid:1 H₂O solution). The column was eluted usingthe same solution and the fractions containing product were combined,concentrated under reduced pressure and axeotropically dried fromheptane. This procedure was repeated a second time before the resultantisolated solids were dissolved in anhydrous THF and loaded onto a silicagel prep plate. The prep plate was eluted once using 66% toulene:14%dioxane: 18% heptane: 4% acetic acid. The upper band was scraped off theprep plate and digested with 10 mL of anhydrous THF, filtered and rinsedwith 20 mL of THF. The filtrate was concentrated in vacuo to afford 59mg of title compound as an orange red residue in 85% AUC HPLC purity. ¹HNMR (300 MHz; Acetone d₆) δ 7.67 (s, 1H), 6.99 (s, 1H), 3.27 (m, 1H),1.12 (d, J=5.4 Hz 3H), 1.06 (d, J=5.1 Hz, 3H). MS MSD Trap: m/z 888.6(M−1)⁻ (exact mass 887.65). UV-VIS λmax=552 nm in methanol.

This description has been offered for illustrative purposes only and isnot intended to limit the invention of this application.

What is claimed as new and desired to be protected by Letters Patent isset forth in the appended claims.

We claim:
 1. A process for the preparation of a compound of Formula 4:

wherein R₁ is independently Cl or Br, R₂, R₃, R₄ and R₅ are I, R₆ is H,and R₁₁ and R₁₂ are independently H or a counter-ion capable of forminga pharmaceutically acceptable salt, comprising: a) combining a compoundof Formula 1a,

wherein R₁ is independently Cl or Br, with about two equivalents of acompound of Formula 2,

wherein R₇, R₈, R₉ and R₁₀ are H, in an acidic solution substantiallyfree of chloride ions and substantially free of chloride-ion producingimpurities, to form an intermediate compound of Formula 3a,

wherein R₁ is independently Cl or Br and R₂, R₃, R₄, R₅ and R₆ are H; b)combining the intermediate compound of Formula 3a with at least 4equivalents of iodine in a solution substantially free of chloride ionsand substantially free of chloride-producing impurities to form thecompound of Formula 4 substantially free of transhalogenated impuritiesof the compound of Formula 4 wherein R₁ is independently Cl or Br; atleast one of R₂, R₃, R₄ and R₅ is Cl and the remainder are I; R₆ is H;and R₁₁ and R₁₂ are independently H, Na, K, Li, or a counter-ion capableof forming a pharmaceutically acceptable salt.
 2. The process of claim 1wherein a transhalogenated impurity of the compound of Formula 4comprises less than 0.15 percent by weight.
 3. The process of claim 1wherein the transhalogenated impurities comprise at least one compoundselected from the group consisting of:2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-Spiro[isobenzofuran-1,9′-xanthen]-3-one;an isomeric quinoid thereof; and mixtures thereof.
 4. The process ofclaim 1 wherein the acidic solution comprises less than 1500 ppmchloride.
 5. The process of claim 1 wherein the acidic solutioncomprises at least one acid selected from the group consisting of analkyl sulfonic acid or aryl sulfonic acid with a melting point of lessthan 250° C., an alkyl carboxylic acid or aryl carboxylic acid with amelting point of less than 250° C., a non-chloride Br{acute over(ø)}nsted acid, a non-chloride Lewis Acid, a polymer bound preparationthereof, a salt thereof, an aqueous solution thereof, and mixturesthereof, alone or in combination with methanesulfonic acid.
 6. Theprocess of claim 1 wherein the acidic solution comprises at least oneacid selected from the group consisting of p-toluenesulfonic acid,benzenesulfonic acid, sulfuric acid, trifluoromethanesulfonic acid,ethanesulfonic acid, acetic acid, propionic acid, trifluoroacetic acid,camphorsulfonic acid, a polymer bound preparation thereof, a saltthereof, an aqueous solution thereof, and mixtures thereof, alone or incombination with methanesulfonic acid.
 7. The process of claim 1 whereinthe acidic solution comprises methanesulfonic acid.
 8. The process ofclaim 1 wherein the compounds of Formula 2 and Formula 1a are combinedin a ratio in the range of 2.5:1 to 3.2:1.
 9. The process of claim 1further comprising heating the compounds of Formula 1a and Formula 2 inthe acidic solution at a temperature in the range of 85° C. to 95° C.for a time period of 1 to 16 hours.
 10. The process of claim 1 whereinthe compound of Formula 1a is in a mixture with Formula 1

and the mixture is combined with two equivalents of the compound ofFormula
 2. 11. The process of claim 1 further comprising isolating theintermediate compound of Formula 3a.
 12. The process of claim 11 whereinthe isolating comprises suspension of the intermediate compound ofFormula 3a, collection of the intermediate compound of Formula 3a, andrinsing the intermediate compound of Formula 3a with a substantiallychloride-ion-free solvent.
 13. The process of claim 12 wherein thesolvent comprises a mixture of acetone and water heated to 60° C. 14.The process of claim 1 wherein the solution containing the intermediatecompound of Formula 3a and iodine comprises less than 1500 ppm chloride.15. The process of claim 1 further comprising heating the combinedintermediate compound of Formula 3a and iodine in basic solution at atemperature in the range of 20° C. to 100° C. for a time period in therange of 1 to 24 hours.
 16. The process of claim 15 further comprisingheating the combined intermediate compound of Formula 3a and iodine insolution at a temperature in the range of 70° C. to 95° C. for a timeperiod in the range of 1 to 24 hours.
 17. The process of claim 1 whereinthe solution comprises at least one base selected from the groupconsisting of sodium hydroxide, potassium hydroxide, lithium hydroxide,sodium bicarbonate, potassium bicarbonate, and mixtures thereof.
 18. Theprocess of claim 1 wherein the solution comprises sodium hydroxide at aconcentration of 0.4 to 1.0 M.
 19. The process of claim 1 furthercomprising adding at least one iodine-solubilizing agent to the solutioncomprising the intermediate compound of Formula 3a and iodine whereinthe iodine-solubilizing agent is selected from the group consisting ofpotassium iodide, lithium iodide, sodium iodide, and mixtures thereof.20. The process of claim 19 wherein the iodine-solubilizing agentcomprises 1 to 2.5 equivalents of sodium iodide.
 21. The process ofclaim 1 wherein iodine is generated in situ by adding to the solution anoxidizing agent and an iodide salt wherein the solution is substantiallyfree of chloride ions, chloride-ion free radicals, hypochlorite,hypochlorous acid or mixtures thereof.
 22. The process of claim 1further comprising adding an iodine scavenger to the solution subsequentto the formation of the compound of Formula 4, wherein the scavenger isselected from the group consisting of sodium thiosulfate, potassiumthiosulfate, ammonium thiosulfate, potassium sulfite, sodium sulfite andmixtures thereof.
 23. The process of claim 22 wherein the iodinescavenger is sodium sulfite.
 24. The process of claim 23 wherein theaddition of the iodine scavenger is performed at a temperature of 10° C.or lower.
 25. The process of claim 1 wherein R₁₁ and R₁₂ are sodium. 26.A process for the preparation of a compound of Formula 3

wherein R₁ is independently Cl or Br, R₂, R₃, R₄ and R₅ are I and R₆ isH, comprising: a) combining a compound of Formula 1a,

wherein R₁ is independently Cl or Br, with two equivalents of a compoundof Formula 2,

wherein R₇, R₈, R₉ and R₁₀ are H, in an acidic solution at a temperaturein the range of 20° C. to 250° C. to form a compound of Formula 3a; b)isolating the intermediate compound of Formula 3a,

wherein R₁ is independently Cl or Br and R₂, R₃, R₄, R₅ and R₆ are H; c)combining the intermediate compound of Formula 3a with 4 equivalents ofiodine in a solution substantially free of chloride ions andsubstantially free of chloride-ion-producing impurities at a temperaturein the range of 20° C. to 100° C. to form the compound of Formula 3; d)adding an iodine scavenger to the compound of Formula 3; e) acidifyingthe compound of Formula 3 to a pH of less than 5 and cooling to atemperature of less than 10° C.; and f) isolating the compound ofFormula 3 substantially free of transhalogenated impurities wherein R₁is independently Cl or Br, at least one of R₂, R₃, R₄ and R₅ is Cl andthe remainder are I, and R₆ is H.
 27. The process of claim 26 wherein atranshalogenated impurity of the compound of Formula 3 comprises lessthan 0.15 percent by weight.
 28. The process of claim 26 wherein thetranshalogenated impurities comprise at least one compound selected fromthe group consisting of:2′,4,5,6,7-pentachloro-3′,6′-dihydroxy-4′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;4,4′,5,6,7-pentachloro-3′,6′-dihydroxy-2′,5′,7′-triiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;2′,4,5,6,7,7′-hexachloro-3′,6′-dihydroxy-4′,5′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;4,4′,5,5′,6,7-hexachloro-3′,6′-dihydroxy-2′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;2′,4,5,5′,6,7-hexachloro-3′,6′-dihydroxy-4′,7′-diiodo-3H-spiro[isobenzofuran-1,9′-xanthen]-3-one;an isomeric quinoid thereof; and mixtures thereof.
 29. The process ofclaim 26 wherein the acidic solution comprises less than 1500 ppmchloride.
 30. The process of claim 26 wherein the acidic solutioncomprises at least one acid selected from the group consisting of analkyl sulfonic acid or aryl sulfonic acid with a melting point of lessthan 250° C., an alkyl carboxylic acid or aryl carboxylic acid with amelting point of less than 250° C., a non-chloride Br{acute over(ø)}nsted acid, a non-chloride Lewis Acid, a polymer bound preparationthereof, a salt thereof, an aqueous solution thereof, and mixturesthereof, alone or in combination with methanesulfonic acid.
 31. Theprocess of claim 26 wherein the acidic solution comprises at least oneacid selected from the group consisting of p-toluenesulfonic acid,benzenesulfonic acid, sulfuric acid, trifluoromethanesulfonic acid,ethanesulfonic acid, acetic acid, propionic acid, trifluoroacetic acid,camphorsulfonic acid, a polymer bound preparation thereof, a saltthereof, an aqueous solution thereof, and mixtures thereof, alone or incombination with methanesulfonic acid.
 32. The process of claim 26wherein the acidic solution comprises methanesulfonic acid.
 33. Theprocess of claim 26 wherein the compounds of Formula 2 and Formula 1aare combined in a ratio in the range of 2.5:1 to 3.2:1.
 34. The processof claim 26 further comprising heating the compounds of Formula 1a andFormula 2 in the acidic solution at a temperature in the range of 85° C.to 95° C. for a time period of 1 to 16 hours.
 35. The process of claim34 wherein the compound of Formula 1a is in a mixture with Formula 1

and the mixture is combined with two equivalents of the compound ofFormula
 2. 36. The process of claim 26 further comprising isolating theintermediate compound of Formula 3a.
 37. The process of claim 36 whereinthe isolating comprises suspension of the intermediate compound ofFormula 3a, collection of the intermediate compound of Formula 3a, andrinsing the intermediate compound of Formula 3a with a substantiallychloride-ion-free solvent.
 38. The process of claim 37 wherein thesolvent comprises a mixture of acetone and water heated to 60° C. 39.The process of claim 26 wherein the solution containing the intermediatecompound of Formula 3a comprises less than 1500 ppm chloride.
 40. Theprocess of claim 26 further comprising heating the combined intermediatecompound of Formula 3a and iodine in basic solution at a temperature inthe range of 20° C. to 100° C. for a time period in the range of 1 to 24hours.
 41. The process of claim 40 further comprising heating theintermediate compound of Formula 3a and iodine in solution at atemperature in the range of 70° C. to 95° C. for a time period in therange of 1 to 24 hours.
 42. The process of claim 26 wherein the solutionof combined intermediate compound of Formula 3a and iodine comprises atleast one base selected from the group consisting of sodium hydroxide,potassium hydroxide, lithium hydroxide, sodium bicarbonate, potassiumbicarbonate, and mixtures thereof.
 43. The process of claim 26 whereinthe solution comprises sodium hydroxide at a concentration of 0.4 to 1.0M.
 44. The process of claim 26 further comprising adding at least oneiodine-solubilizing agent to the solution comprising the combinedintermediate compound of Formula 3a and iodine wherein theiodine-solubilizing agent is selected from the group consisting ofpotassium iodide, lithium iodide, sodium iodide, and mixtures thereof.45. The process of claim 44 wherein the iodine-solubilizing agentcomprises 1 to 2.5 equivalents of sodium iodide.
 46. The process ofclaim 26 wherein iodine is generated in situ by adding to the solutioncontaining the intermediate compound of Formula 3a an oxidizing agentand an iodide salt wherein the solution is substantially free ofchloride ions, chloride-ion free radicals, hypochlorite, hypochlorousacid or mixtures thereof.
 47. The process of claim 26 further comprisingadding an iodine scavenger to the solution subsequent to the formationof the compound of Formula 3, wherein the scavenger is selected from thegroup consisting of sodium thiosulfate, potassium thiosulfate, ammoniumthiosulfate, potassium sulfite, sodium sulfite and mixtures thereof. 48.The process of claim 47 wherein the iodine scavenger is sodium sulfite.49. The process of claim 48 wherein the addition of iodine scavenger isperformed at a temperature of 10° C. or lower.
 50. The process of claim26 wherein acidifying comprises adding a chloride-ion-free acid to thesolution containing Formula
 3. 51. The process of claim 50 wherein thechloride-ion-free acid comprises sulfuric acid at a concentration of 1percent to 5 percent, and where this sulfuric acid is added insufficient quantity to adjust pH of the solution to be in the range ofpH 2 to pH
 5. 52. The process of claim 51 wherein the pH is adjusted toapproximately
 3. 53. The process of claim 26 wherein the isolatingcomprises suspending the compound of Formula 3 in a solvent selectedfrom the group consisting of water, dimethylformamide, acetone andmixtures thereof; vacuum filtration; and rinsing the resultant filtercake with a solvent selected from the group consisting of water,dimethylformamide, acetone and mixtures thereof.
 54. The process ofclaim 26 wherein said compound of Formula 3, in which R₁ isindependently Cl or Br, R₂, R₃, R₄, R₅ are I and R₆ is H, is convertedto the quinoid form of Formula 4, wherein R₁₁ and R₁₂ are independentlyH or a counter-ion capable of forming a pharmaceutically acceptable salt


55. The process of claim 54 wherein R₁₁ and R₁₂ are sodium.